ES2295858T3 - EM MUTATIONS THE HUMAN PCSK9 GEN ASSOCIATED WITH HYPERCHOLESTEROLEMIA. - Google Patents

Abstract

Procedure for detecting the presence of or predisposition to hypercholesterolemia, disorders of lipid and lipoprotein metabolism, atherosclerosis or cardiovascular diseases in a subject, the method comprising detecting in a sample of said subject the presence of an alteration in the gene PCSK9 or in the NARC-1 polypeptide, said alteration in said gene or said polypeptide being indicative of the presence of or predisposition to hypercholesterolemia, disorders of lipid and lipoprotein metabolism, atherosclerosis or cardiovascular diseases and being selected from among the group consisting of a TA substitution in nucleotide 625 of SEQ. ID. No. 1, a T-C substitution in nucleotide 890 of SEQ. ID. No. 1, an A-G substitution in nucleotide 19697 of SEQ. ID. nº: 3, a substitution of the serine residue in position 127 of the SEC. ID. No. 2 with an arginine, a substitution of the phenylalanine residue at position 216 of the SEC. ID. No. 2 with a leucine, a substitution of the isoleucine moiety at position 474 of the SEC. ID. No. 2 for a valine and a combination thereof.

Description

Mutations in the human PCSK9 gene associated with hypercholesterolemia.

Introduction

The present invention relates generally to the fields of genetics and medicine. The present invention more specifically discloses the identification of a gene causing human hypercholesterolemia, which can be used for the diagnosis, prevention and treatment of hypercholesterolemia, and more specifically of familial ADH hypercholesterolemia, as well as for the identification of therapeutically active drugs. The invention discloses more specifically that mutations in the PCSK9 gene encoding NARC-1 produce autosomal dominant hypercholesterolemia (ADH) and represent new targets for therapeutic intervention. The invention can be used for the diagnosis of predisposition to the detection, prevention and / or treatment of disorders of cholesterol and lipoprotein metabolism, including known hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis and more generally cardiovascular diseases (CVD).

Background

Atherosclerosis is a disease of the arteries responsible for coronary heart disease (CVD) that is responsible for the majority of deaths in industrialized countries (Lusis, 2000). Several risk factors for CHD have now been correctly determined: dyslipidemias, hypertension, diabetes, smoking, poor diet, inactivity and stress. The most clinically relevant and common dyslipidemias are characterized by an increase in beta-lipoproteins (VLDL and LDL particles) with hypercholesterolemia in the absence or presence of hypertriglyceridemia (Fredrickson et al ., 1967). An isolated elevation of LDL cholesterol is one of the most frequent risk factors for CVD. Duplicate studies (Austin et al ., 1987) and known data (Perusse, 1989; Rice et al ., 1991) have demonstrated the importance of genetic factors in the development of the disease, particularly when complications occur early in life . Mendelian forms of hypercholesterolemia have been identified: first the autosomal dominant form (ADH) (Khachadurian, 1964) and then the autosomal recessive form (ARH), initially described as "pseudohomozyotic type II hyperlipoproteinemia" (Morganroth et al ., 1967 ).

ADH is a heterogeneous genetic disorder. Its most frequent and archetypal form is familial hypercholesterolemia (FH) with a frequency of 1 in 500 for heterozygotes and 1 per million for homozygous (Goldstein et al ., 1973). The disease is codominant with homozygotes that are initially affected and more severely than heterozygotes. FH is produced by mutations in the gene encoding the LDL receptor (Goldstein and Brown, 1978) ( LDLR in 19p13.1-p13.3) (MIM 143890). It is characterized in that it presents a selective increase in plasma LDL cholesterol concentrations that give rise to tendon and skin xanthomas, corneal arch and cardiovascular deposits that lead to progressive and premature atherosclerosis, CHD and mortality ( which occurs before age 55). The second form of ADH is familial defective apo B-100 (FDB) produced by mutations in the apolipoprotein B gene ( APOB in 2p23-p24), which encodes the LDL receptor ligand (Inneraty et al ., 1987) ( MIM 144010). The existence of a higher level of genetic heterogeneity in ADH has been described (Saint-Jore et al . 2000) and the implication of a third locus called HCHOLA3 (formerly FH3 ) in 1p34.1 p32 in a French family was detected and mapped ( Varret et al ., 1999) (MIM 603776). These results were confirmed by Hunt et al . in a large family of Utah (Hunt et al ., 2000).

There is a great need to identify genes involved in hypercholesterolemia, more specifically in ADH; in order to understand the mechanisms that lead to these disorders and develop diagnostic and therapeutic treatments improved

Summary of the invention

The inventors have shown that mutations in the PCSK9 gene encoding NARC-1 produce autosomal dominant hypercholesterolemia. They have shown that NARC-1 protein contributes to cholesterol homeostasis. The invention thus describes new targets for diagnosis and therapeutic intervention aimed at hypercholesterolemia, more specifically ADH, CVD, disorders of lipid and lipoprotein metabolism, atherogenic dyslipidemia, atherosclerosis and cardiovascular diseases.

In a first aspect the invention describes a PCSK9 gene or a fragment thereof comprising an alteration, reducing said alteration, modifying or suppressing the activity of NARC-1. Preferably, said alteration is a nucleotide substitution. More preferably, said nucleotide substitution leads to an amino acid change in the NARC-1 protein. Preferably, said amino acid change is located at or near the catalytic zone or a zymogen preparation of the NARC-1 protein and decreases the catalytic activity or the autocatalytic cleavage of said protein or functional domain, respectively. Alternatively, the alteration affects the splicing of the NARC-1 mRNA.

The invention also describes a protein. Corresponding NARC-1 or a fragment thereof which comprises an alteration, reducing said alteration, modifying or suppressing the activity of NARC-1. Preferably, the alteration is located in the catalytic zone of the NARC-1 protein and decreases its activity catalytic or in a zymogen making zone of NARC-1 and decreases its autocatalytic excision. In another preferred embodiment, the alteration is located next to the catalytic zone of the NARC-1 protein and decreases its catalytic activity or close to the areas of elaboration of NARC-1 zymogen and decreases its autocatalytic excision.

One aspect of the present invention describes a genotyping method in a subject a polymorphism of the PCSK9 gene, preferably a polymorphism described in Table 2. The invention also relates to an association procedure of one or more polymorphism (s) of the PCSK9 gene , preferably one or more polymorphism (s) described in Table 2 for a disease or disorder.

Another aspect of the present invention describes a method of detecting the presence of hypercholesterolemia or its predisposition, more specifically ADH or disorders of lipid and lipoprotein metabolism in a subject, the method comprising detection in a sample of the subject of the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, the presence of said alteration indicating the presence or predisposition to hypercholesterolemia, more specifically ADH or lipid and lipoprotein metabolism disorders. In a more preferred embodiment, said alteration reduces, modifies or suppresses the activity of NARC-1. Optionally, the method further comprises detecting the presence of an alteration in the LDL receptor and / or apolipoprotein B in said sample.

The invention also describes a diagnostic kit comprising primers, probes and / or antibodies for detecting in a sample of a subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, in the NARC-1 RNA or the expression of the polypeptide and / or the activity of NARC-1. Optionally, said diagnostic kit further comprises reagents for detecting in a sample of a subject the presence of an alteration in the LDL receptor and / or apolipoprotein B.

Another aspect of the invention describes the use of a functional NARC-1, preferably a natural NARC-1 protein or a nucleic acid that encodes it, for the preparation, of a composition pharmaceutical intended to treat or prevent hypercholesterolemia, more specifically ADH and / or metabolic disorders of lipids and lipoproteins in a subject. The invention relates also to the use of a biologically active compound that modulates the activity of NARC-1, for the preparation of a pharmaceutical composition intended to treat or prevent hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism in a subject. The invention also describes a method for treating or preventing hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism in a subject comprising the administration to said subject of a Functional NARC-1, preferably a protein Natural NARC-1 or a nucleic acid encoding the same. The invention further describes a method for treating or prevent hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism in a subject comprising administering to said subject a biologically compound  active that modulates the activity of NARC-1.

Another aspect of the present invention gives know methods of selection of biologically active compounds that modulate the activity of the NARC-1 protein, by general rule of an altered NARC-1 polypeptide. The compounds are more specifically suitable for treating the hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism.

Legends of the figures Figure 1 Genealogical and genetic analysis of the HC92 family with markers covering zone 1p34.1-p32

Affected subjects have a history of tendon xanthomas (HC92-II-7 and III-3), CHD, early myocardial infarction (HC92-II-2 and II-6) and stroke (HC92-D-4). The allele Affected is represented by black bars. The age (in years) in the measurement of lipids, total cholesterol and LDL (in g / l; untreated values for affected members).

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Figure 2 Genetic analysis of the HC2 family

The genealogy of the HC2 family is shown. The symbols with the black half indicate the affected members, the non-blackened symbols indicate unaffected members and raster symbols indicate the members with a phenotypic state known. The haplotype in parentheses of the subject HC2-I-1 was unequivocally deduced. Selected markers that comprise the zone 1p34.1-p32 are presented to the left of the genealogies The affected allele is represented by the bars black

Age (in years) is provided in the measurement of lipids, total cholesterol and LDL (in g / l; untreated values for affected members).

Figure 3 Genetic analysis and mutation detection in families HC92 and HC60

a , Results of LINKMAP analyzes in the HC92 family that indicate a maximum Iod score for D1S2742 at the = 0. PCSK9 1.2 Mb cartography of this marker. b , Mutation in the HC92 family. The test (HC92-II-7) is heterozygous for a T → substitution in exon 2 in nucleotide 625 (S127R). c , Family genealogical and genetic analysis of the HC60 family. d , Analysis of the sequence in the HC60 family. Test (HC60-II-2) is heterozygous for a T → C substitution in exon 4 in nucleotide 890 that predicts a leucine 216 substitution by conserved phenylalanine (F216L).

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Figure 4 Mutation Study

a , Segregation of the S127R mutation in part of the HC2 family. Substitution T → in nucleotide 625 creates a new recognition cleavage zone for restriction digestion by Mn / l (represented by *). After electrophoretic migration on a 2% agarose gel, 208, 203 and 60 base pair fragments were distinguished in the normal allele, while 208, 143 and 60 base pair fragments appeared in the mutated alleles (The normal 203 base pair fragment was divided into 143 and 60 base pair fragments and the two 60 base pair fragments generated migrated together). The test (HC2-II-9) and one of his children (HC2-III-10) were found to be heterozygous for the S127R mutation (as indicated by both bands of 203 and 143 bp).

b , The alignment of the amino acid sequence for NARC-1 shows serine conservation in codon 127 between man, mouse and rat. DNA sequences for normal and mutant genes are presented above and below amino acid sequences, respectively.

c , The alignment of the amino acid sequence for NARC-1 presents phenylalanine conservation in codon 216 between man, mouse and rat. DNA sequences for normal and mutant genes are presented above and below amino acid sequences, respectively.

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Detailed description of the invention Definition

The PCSK9 gene (or NARC-1 gene) encodes the NARC-1 protein or polypeptide. The NARC-1 protein is translated as a preprotein that is processed autocatalytically in a mature NARC-1 protein. The sequence of the NARC-1 gene has been described in patent applications WO 01/57081 and WO 02/14358 and partially characterized in Seidah et al . (2003). The remains of the catalytic zone of NARC-1 consist of Asp-186, Ser-188, His-226, Asn-317 and Ser-386. NARC-1 has two zones of zymogen production: a first one comprising residues 78 to 82 and having a primary cleavage zone located at position 82. A second one comprising residues 138 and 142 and having a cleavage zone secondary status at position 142. The biological function of NARC-1 and the involvement of this protein in hypercholesterolemia and disorders of lipid and lipoprotein metabolism were unknown.

Within the context of the present invention, the locus of the PCSK9 gene designates all sequences or products of PCSK9 in a cell or organism, including coding sequences of PCSK9 , non-coding sequences of PCSK9 (e.g., introns, 5 'and 3' UTR), the regulatory sequences of PCSK9 that control transcription and / or translation (e.g., activator, enhancer, terminator, etc.), as well as all corresponding expression products, such as RNAs of PCSK9 (eg mRNA) and NARC-1 polypeptides (eg, a preprotein and a mature protein).

The term "gene" shall be considered to include any type of nucleic acid encoding, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any corresponding form of RNA. The term gene specifically includes recombinant nucleic acids encoding NARC-1, that is, any artificially created non-natural nucleic acid molecule, e.g. eg, assembling, cutting, linking or expanding the sequences. A PCSK9 gene is as a rule double-stranded, although other forms, such as single-stranded, can be contemplated. PCSK9 genes can be obtained from various sources and according to various techniques known in the art, such as DNA screening banks or by extension of various natural sources. Recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques or a combination thereof. A specific example of a PCSK9 gene comprises SEC. ID. nº: 1.

The positions indicated in a PCSK9 gene and an NARC-1 protein refer to the positions in the SEC sequences. ID. nº: 1 and SEC. ID. nº: 2, respectively.

The expression "hybridizes under severe conditions" means that two nucleic acid fragments can hybridize with one another under usual hybridization conditions described in Sambrook et al ., Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, New York , US. More specifically, "severe conditions" as used herein refer to hybridization at 65 ° C in a hybridization buffer consisting of 250 mmol / l of sodium phosphate buffer pH 7.2, 7% SDS (w / v), 1% BSA (w / v), 1 mmol / l EDTA and 0.1 mg / ml single stranded salmon sperm DNA.

Genes and proteins

The invention describes an isolated or recombinant PCSK9 gene comprising an alteration that produces hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism.

The altered PCSK9 gene comprises an alteration that leads to a decrease or a complete loss of NARC-1 activity, or a new NARC-1 activity. This decrease, loss or new activity may be due to a decrease or loss of the activity of the NARC-1 enzyme, to a decrease in the stability of NARC-1 (one of the two at the stage of nucleic acid, preprotein or protein ), to a change in substrate specificity of NARC-1 or to the alteration or impediment of the polymerization of NARC-1. This decrease or loss of NARC-1 activity may be due to an alteration of NARC-1 that leads to a decrease or loss of maturation of pro-NARC-1, one of both at the first excision or the second or both. The alteration may also affect the catalytic activity by modifying the catalytic zone of NARC-1 or its substrate recognition zone. In addition, the alteration may affect the splicing of the NARC-1 mRNA, which leads to an alternative splicing product.

The invention describes an isolated or recombinant PCSK9 gene or a fragment thereof comprising an alteration, wherein said alteration reduces, modifies or suppresses the activity of NARC-1. Preferably, said alteration is a nucleotide substitution. More preferably, said nucleotide substitution leads to a change of amino acids in the NARC-1 protein. Preferably, said amino acid change is located in (for example, within) the catalytic zone of the NARC-1 protein and decreases its catalytic activity, or is located in a zymogen manufacturing zone of NARC-1 and decreases its autocatalytic cleavage . In another preferred embodiment, said amino acid change is located close to the catalytic zone of the NARC-1 protein and decreases its catalytic activity or close to the zymogen manufacturing zones of NARC-1 and decreases its autocatalytic cleavage. Alternatively, the interaction may affect the splicing of NARC-1 mRNA. More specifically, said alteration may be a substitution at nucleotide 625 and / or 890 of the SEC. ID. No .: 1. More preferably, said alteration is selected from the group consisting of a T → substitution in nucleotide 625 of the SEC. ID. No. 1, a substitution T → C in nucleotide 890 of SEQ. ID. nº: 1 and a combination thereof. In another embodiment, said alteration is selected from the group consisting of a substitution at nucleotides 476-478 of the SEC. ID. No. 1, a substitution in nucleotides 482-484 of SEQ. ID. No. 1, a substitution in nucleotides 488-490 of SEQ. ID. No. 1, a substitution in nucleotides 485-490 of SEQ. ID. No. 1, a substitution in nucleotides 548-553 of SEC. ID. No. 1, a substitution in nucleotides 479-481, 491-493 and 578-580 of SEQ. ID. No. 1, a substitution at nucleotides 620-622 of SEQ. ID. No. 1, a substitution at nucleotides 656-658 of SEQ. ID. No. 1, a substitution at nucleotides 671-673 of SEC. ID. No. 1, a substitution in nucleotides 920-922 of SEC. ID. No. 1 and a substitution at nucleotides 1193-1195 of SEC. ID. nº: 1.

The invention also describes an isolated or recombinant PCSK9 gene or a fragment thereof comprising at least one alteration, wherein said alteration is selected from the group consisting of the polymorphisms listed in Table 2 and in Table 4.

The present invention relates to an isolated or recombinant PCSK9 gene or a fragment thereof as defined in claim 5.

The invention describes a protein NARC-1 isolated or purified or a fragment of the same that comprises an alteration, in which said alteration reduce, modify or suppress the activity of NARC-1. Preferably, the alteration is located in the catalytic zone of the NARC-1 protein and decreases its activity catalytic or in a zymogen making zone of NARC-1 and decreases its catalytic activity. In other preferred embodiment, the alteration is located next to the catalytic zone of the NARC-1 protein and decreases its catalytic activity or close to the areas of elaboration of NARC-1 zymogen and decreases its autocatalytic excision. More preferably, said alteration can be selected from the group consisting of a replacement of the serine residue at position 127, a substitution of the phenylalanine residue at position 216 and a combination of the same. Even more preferably, said alteration is selected from between the group constituted by a substitution of the serine residue in SEC position 127. ID. No .: 2 for an arginine (S127R), a replacement of the phenylalanine residue at position 216 of the SEC. ID. nº: 2 for a leucine (F216L) and a combination thereof. In another embodiment, said alteration is selected from between the group consisting of a substitution of the tyrosine residue in position 78 of the SEC. ID. nº: 2, a substitution of the rest valine at position 80 of the SEC. ID. nº: 2, a substitution of leucine residue at position 82 of the SEC. ID. nº: 2, one replacement of valine residues at positions 79, 80 and 81 of the SEC. ID. nº: 2, a substitution of the remains of alanines in the positions 102 and 103 of the SEC. ID. nº: 2, a substitution of Valine residues at position 79, lysine at position 83 and of leucine at position 112 of the SEC. ID. nº: 2, a substitution of the rest of methionine at position 126 of the SEC. ID. nº: 2, one replacement of the proline residue at position 138 of the SEC. ID. : 2, a substitution of the isoleucine residue at position 143 of the SEC. ID. nº: 2, a replacement of the histidine residue in the position 226 of SEC. ID. nº: 2 and a replacement for the rest of asparagine in position 317 of the SEC. ID. nº: 2. Preferably, said alteration is selected from the group consisting of a replacement of the tyrosine residue at position 78 of the SEC. ID. : 2 for an alanine (Y78A), a replacement for the rest of valine in the SEC position 80. ID. nº: 2 for an alanine or a leucine (V80A or V80L), a replacement for the leucine residue at position 82 of the SEC. ID. nº: 2 for an alanine, a valine or a proline (L82A, L82V or L82P), a replacement for valine residues in positions 79, 80 and 81 of the SEC. ID. nº: 2 for an arginine, one arginine and a leucine, respectively (V79R, V80R and V81L), a replacement of the remains of alanines in positions 102 and 103 of the SEC. ID. nº: 2 by arginines (A102R and A103R), a substitution of the remains of valine at position 79, lysine at position 83 and leucine at position 112 of the SEC. ID. nº: 2 for one isoleucine, a methionine, a proline, respectively (V79I, K83M and L112P), a substitution of the methionine residue at position 126 of the SEC. ID. nº: 2 by an alanine (M126A), a substitution of proline residue at position 138 of the SEC. ID. nº: 2 for one tyrosine (P138Y), a replacement for the rest of isoleucine in the SEC position 143. ID. nº: 2 for a proline (I143P), a replacement of histidine moiety at position 226 of SEC. ID. nº: 2 with an alanine (H226A) and a substitution of the rest of asparagine in position 317 of the SEC. ID. nº: 2 for an alanine (N317A). Alternatively, said alteration may be selected from between the group constituted by a substitution of the rest of arginine at position 218 of the SEC. ID. nº: 2, a substitution of the rest of arginine at position 237 of the SEC. ID. nº: 2 and one combination thereof, more preferably a replacement of the arginine residue at position 218 of the SEC. ID. nº: 2 for one serine (R218S) or a substitution of the rest of arginine in the position 237 of the SEC. ID. nº: 2 for a tryptophan (R237W) or a combination thereof.

The invention also describes a protein. NARC-1 isolated or purified or a fragment of the which comprises an alteration, in which said alteration is select from the group consisting of an insertion of a leucine residue in position 15 of the SEC. ID. nº: 2, one replacement of the rest of arginine at position 46 of the SEC. ID. nº: 2 for a leucine (R46L), a substitution of the rest of alanine in position 53 of the SEC. ID. nº: 2 for a valine (A53V), a replacement of the rest of leucine at position 474 of the SEC. ID. nº: 2 for a valine (I474V), a substitution of the rest of acid glutamic at position 670 of SEC. ID. nº: 2 for a glycine (E670G) and a combination thereof. The invention also describes an isolated or purified NARC-1 protein or a fragment thereof comprising an alteration described in the Table 4

The present invention relates to a protein NARC-1 isolated or purified or to a fragment of the same as defined in claims 6 and 7.

The invention further describes a nucleic acid. recombinant encoding an NARC-1 protein or a fragment thereof comprising an alteration according to the present invention, a vector comprising said nucleic acid, a host cell comprising said vector or said acid recombinant nucleic and a non-human host organism that comprises said recombinant nucleic acid, said vector or said host cell

Therefore, the invention describes an isolated or recombinant PCSK9 gene and / or the NARC-1 protein comprising an alteration that produces hypercholesterolemia, more specifically ADH, reducing said alteration, modifying or suppressing the activity of NARC-1. In this context, modifying means a change in specificity of the NARC-1 protein. Optionally said alteration decreases or suppresses the stability of the NARC-1 protein. Optionally, said alteration decreases or suppresses the stability of the mRNA encoding NARC-1. Optionally, said alteration reduces the transcription rate of the PCSK9 gene. Optionally, said alteration decreases or suppresses the activity of the NARC-1 protein. Optionally, said alteration decreases or suppresses the specificity of NARC-1 for at least one of its natural substrates. Optionally, said alteration introduces a new specificity of NARC-1 for an uncommon substrate. Said infrequent substrate is preferably involved in the metabolism of cholesterol and / or lipoproteins. Optionally, said alteration prevents or prevents the polymerization of NARC-1. Optionally, said alteration affects the catalytic zone of NARC-1. Optionally, said alteration affects the recognition zone of the NARC-1 substrate. Optionally, said alteration affects the development of pro-NARC-1 in NARC-1. More particularly, said alteration reduces or prevents autocatalytic cleavage in one of the two zones of the zymogen or in both zones of the zymogen. Optionally, said alteration modifies the association between NARC-1 and its prosegment, for example by increasing or decreasing its interaction.

By "decrease", it is understood within the context of the present invention that the parameter evaluated be between 10% and 90% of the value of the parameter with a Natural NARC-1 protein in a natural environment. Plus preferably, said evaluated parameter is comprised between the 25% and 75% of the parameter value with a protein Natural NARC-1 in a natural environment. By "delete" is understood within the context of this invention that the evaluated parameter is less than 10% of the value of the parameter with a natural NARC-1 protein in a natural environment More preferably, said evaluated parameter is less than 5% of the parameter value with a protein Natural NARC-1 in a natural environment. Even more preferably, said evaluated parameter is less than 1% of parameter value with a natural NARC-1 protein In a natural environment.

In a specific embodiment, said alteration decreases or suppresses the catalytic activity of NARC-1 Preferably, said alteration is located next to the catalytic zone of the protein NARC-1 Preferably, this alteration is located next to a rest of the selected catalytic zone of between the group consisting of aspartic acid at position 186, serine at position 188, histidine at position 226, asparagine at position 317 and serine at position 386. More preferably, this alteration is located near histidine in the position 226. Alternatively, said alteration may be located in one or several remains of the catalytic zone selected from the group constituted by aspartic acid in position 186, serine in the position 188, histidine in position 226, asparagine in the position 317 and serine at position 386.

In another preferred embodiment, said alteration decreases the cleavage in an autocatalytic manner of the NARC-1 protein. Preferably, said alteration It is located close to the zymogen manufacturing areas of NARC-1 These zymogen manufacturing zones They are located at positions 78 to 82 and 138 to 142. Alternatively, said alteration may be located in one or several remains of the zymogen manufacturing areas of NARC-1

From the point of view of the sequence of amino acids, the term "next" designates, within the context of the present invention, an alteration located less than 90 amino acids, preferably 60 to 30 amino acids, more preferably of 20 amino acids, from a rest of the catalytic zone or the zymogen manufacturing zone. It is also understood that the term "next" does not include remains that are part of the area catalytic or zymogen manufacturing zone, defined in the present invention

From the point of view of the sequence nucleotide, the term "next" indicates that the alteration is located less than 270 nucleotides, preferably 180 to 90 nucleotides, more preferably 60 nucleotides, of a nucleotide comprised in a codon that encodes a remainder of the catalytic zone or from the zymogen manufacturing zone. Said alteration preferably change the codon, thus changing the amino acid in this position in the protein sequence.

In a specific embodiment, the invention describes an isolated or recombinant PCSK9 gene and / or an isolated or purified NARC-1 protein comprising an alteration, said alteration being preferably located in the following positions: 1-30, 32-66, 68-77, 83-225, 227-532 and 534-692 of the SEC. ID. nº: 2.

Such alteration of the PCSK9 gene may be a mutation (eg, a nucleotide substitution), a deletion or an addition of at least one nucleotide. Preferably, said alteration is a point mutation. More preferably, said mutation is selected from the group consisting of a nucleotide substitution at position 625 and / or 890. More preferably, said mutation is selected from the group consisting of a T → substitution at nucleotide 625 of the SEC. ID. No. 1, a substitution T → C in nucleotide 890 of SEQ. ID. nº: 1 and a combination thereof. In this regard, a specific object of the invention relates to a polynucleotide sequence of the SEC. ID. No .: 1 or a polynucleotide comprising a fragment of SEQ. ID. No .: 1 of at least 15 consecutive nucleotides, said polynucleotide comprising either nucleotide A at position 625 or nucleotide C at position 890 or a combination thereof. Another specific object of the present invention relates to a polynucleotide sequence of the SEC. ID. No .: 3 or a polynucleotide comprising a fragment of the SEC. ID. No .: 3 of at least 15 consecutive nucleotides, said polynucleotide comprising either nucleotide A at position 5158 or nucleotide C at position 13539 or a combination thereof.

A fragment of a PCSK9 gene designates any part of at least about 8 consecutive nucleotides of a sequence as described above, preferably at least about 15, more preferably at least about 20 nucleotides, more preferably of at least 30 nucleotides. . The fragments include all possible nucleotide lengths between 80 and 100 nucleotides, preferably between 15 and 100, more preferably between 20 and 100. Said fragment can be useful as a primer or probe to identify an alteration of the PCSK9 gene in a sample of a subject or for genotyping a PCSK9 polymorphism, preferably a polymorphism described in Table 2. Said fragment may be a reagent of a diagnostic kit.

Protein alteration NARC-1 can be a substitution, a deletion or an addition of at least one amino acid. Preferably said alteration is a substitution, more preferably, said substitution is selected from the group consisting of a replacement of the serine residue at position 127 of the SEC. ID. : 2, a substitution of the phenylalanine residue at position 216 of the SEC. ID. nº: 2 and a combination thereof. Even more preferably, said substitution is selected from the group constituted by a substitution of the serine residue at position 127 of the SEC. ID. nº: 2 by an arginine (S127R), a substitution of phenylalanine residue at position 216 of the SEC. ID. nº: 2 by a leucine (F216L) and a combination thereof. To this In this regard, a specific objective of the invention relates to a SEC polypeptide sequence. ID. No .: 2 or a polypeptide that comprises a fragment of the SEC. ID. nº: 2 of at least 8 consecutive amino acids, said polypeptide comprising well the rest of arginine at position 127 or the rest of leucine at position 216 or a combination thereof. The invention describes also a polynucleotide encoding said protein NARC-1 altered.

A fragment of the protein NARC-1 designates any portion of at least approximately 8 consecutive amino acids of a sequence as described above, preferably at least about 15, more preferably at least about 20 amino acids, more preferably of at least 30 amino acids The fragments include all possible nucleotide lengths between 8 and 100 amino acids, preferably between 15 and 100, more preferably between 20 and 100. Said fragment may be useful for preparing antibodies.

The invention also describes an antibody. specific for a NARC-1 protein comprising a alteration according to the present invention. In one embodiment preferred, said alteration produces hypercholesterolemia, more particularly ADH and / or disorders of lipid metabolism and / or lipoprotein More preferably, the invention describes a specific antibody of an NARC-1 protein that it comprises a substitution of the serine residue at position 127 of the SEC. ID. nº: 2 by an arginine (S127R) or a substitution of the rest of phenylalanine at position 216 of the SEC. ID. nº: 2 for one leucine (F216L) or a combination thereof. Besides, the invention describes a protein specific antibody NARC-1 comprising a selected alteration of between the group consisting of an insert of a leucine residue in position 15 of the SEC. ID. nº: 2, a substitution of the rest arginine at position 46 of the SEC. ID. nº: 2 for a leucine (R46L), a substitution of the alanine residue at position 53 of the SEC. ID. nº: 2 for a valine (A53V), a replacement for the rest isoleucine at position 474 of the SEC. ID. nº: 2 for a valine (I474V), a substitution of the rest of glutamic acid in the SEC position 670. ID. nº: 2 for a glycine (E670G) and a combination thereof. In addition, the invention describes a specific antibody of an NARC-1 protein that it comprises an alteration described in Table 4, preferably selected from the group consisting of a substitution of tyrosine residue at position 78 of the SEC. ID. nº: 2, one replacement of the remaining valine at position 80 of the SEC. ID. nº: 2, a substitution of the rest of leucine in position 82 of the SEC. ID. nº: 2, a replacement of the valine residues in the positions 79, 80 and 81 of the SEC. ID. nº: 2, a substitution of remains of alanines in positions 102 and 103 of the SEC. ID. nº: 2, a replacement of the valine residues at position 79, lysine at position 83 and leucine at position 112 of the SEC. ID. nº: 2, a substitution of the rest of methionine at position 126 of the SEC. ID. nº: 2, a replacement of the remaining proline in position 138 of SEC. ID. nº: 2, a substitution of the rest of isoleucine in SEC position 143. ID. nº: 2, a substitution of the rest of histidine at position 226 of the SEC. ID. nº: 2 and a substitution of the rest of asparagine in position 317 of the SEC. ID. nº: 2. More preferably, said alteration is selected from the group constituted by a substitution of the rest of tyrosine in the position 78 of SEC. ID. No .: 2 for alanine (Y78A), a replacement for valine residue in position 80 of the SEC. ID. nº: 2 for one valine or a leucine (V80A or V80L), a replacement for the rest of leucine at position 82 of the SEC. ID. nº: 2 for an alanine, one valine or proline (L82A, L82V or L82P), a replacement for Valine residues at positions 79, 80 and 81 of the SEC. ID. nº: 2 for an arginine, an arginine and a leucine, respectively (V79R, V80R and V81L), a replacement for the remains of alanines in positions 102 and 103 of the SEC. ID. nº: 2 for arginines (A102R and A103R), a replacement of the valine residues in the position 79, lysine at position 83 and leucine at position 112 of the SEC. ID. nº: 2 for an isoleucine, a methionine, a proline, respectively (V79I, K83M and L112P), a substitution of the rest of methionine at position 126 of the SEC. ID. nº: 2 for an alanine (M126A), a substitution of the proline residue at position 138 of the SEC. ID. No .: 2 for a tyrosine (P138Y), a replacement for isoleucine residue at position 143 of the SEC. ID. nº: 2 for one proline (I143P), a replacement for the rest of histidine in the position 226 of the SEC. ID. nº: 2 for a alanine (H226A) and a replacement of the remaining asparagine in position 317 of the SEC. ID. No .: 2 for an alanine (N317A). "Specific" means which specifically binds the altered polypeptide and essentially that does not specifically bind the natural polypeptide or the binding of The two ways can discriminate.

An altered PCSK9 gene is also described which has at least one nucleotide mutation at the position included in Table 2. More specifically, the invention describes an altered PCSK9 gene that exhibits the modification of leucine elongation, the corresponding encoded NARC-1 protein. and the use of it.

The present invention discloses new products for use in diagnosis, therapy or identification of hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism. These products comprise nucleic acid molecules that encode a NARC-1 polypeptide according to the present invention, vectors comprising the same, recombinant host cells and expressed polypeptides.

The present invention discloses a vector that comprises a nucleic acid encoding a polypeptide NARC-1 comprising an alteration according to the present invention The vector can be a cloning vector or, more preferably, an expression vector, that is, a vector comprising regulatory sequences that produce the expression of a NARC-1 polypeptide of said vector in a cell competent host.

These vectors can be used to express a NARC-1 polypeptide according to the present invention in vitro , ex vivo or in vivo , to create non-human transgenic or "genetically modified" animals, to amplify nucleic acids, to express complementary RNAs, etc.

The vectors of the present invention typically comprise a sequence encoding NARC-1 according to the present invention functionally linked to regulatory sequences, e.g. eg, an activator, a polyA, etc. The term "functionally linked" indicates that the coding and regulatory sequences are functionally associated so that the regulatory sequences produce the expression (eg, transcription) of the coding sequences. The vectors may further comprise one or more sources of replication and / or selectable markers. The activating zone can be homologous or heterologous with respect to the coding sequence, and provides the ubiquitous, constitutive, regulated and / or tissue-specific expression, in any appropriate host cell, including for use in vivo . Examples of activators include bacterial activators (T7, pTAC, Trp activator, etc.), viral activators (LTR, TK, CMV-IE, etc.), mammalian gene activators (albumin, PGK, etc.) and the like.

The vector can be a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. Plasmid vectors can be prepared from commercially available vectors such as pBluescript, pUC, pBR, etc. Viral vectors can be produced from baculovirus, retrovirus, adenovirus, AAV, etc., according to recombinant DNA techniques known in the matter.

In this regard, the present invention describes a recombinant virus that encodes a polypeptide NARC-1 altered according to the present invention. He Recombinant virus is preferably defective replication, even more preferably selected from among the adenoviruses defective in E1 and / or E4, defective retroviruses in Gag, pol and / or env  and defective AAVs in Rep and Cap. Such recombinant viruses can be produced by techniques known in the art, such as concentrate of transfectant cells or by transfection temporary with plasmids or collaborating viruses. Typical examples of virus cell concentrate include PA317 cells, cells PsiCRIP, GPenv + cells, 293 cells, etc. The detailed protocols to produce said recombinant replication defective viruses they can be found for example in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US No. 5,278,056 and WO 94/19478.

The present invention describes a recombinant host cell comprising a recombinant PCSK9 gene according to the present invention or a vector as defined above. Suitable host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli , Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or created mammalian cell cultures (e.g. eg, produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). More specifically, the invention contemplates the liver and / or the small intestine and the cells thereof or from them.

The present invention also discloses a method for producing a recombinant host cell that expresses an NARC-1 polypeptide comprising an alteration according to the present invention, said method comprising (i) introducing an acid in vitro or ex vivo into a competent host cell Recombinant nucleic or a vector as described above, (ii) culturing in vitro or ex vivo the obtained recombinant host cells and (iii), optionally, selecting the cells that express and / or secrete said NARC-1 polypeptide.

Such recombinant host cells can be used for the production of polypeptides of NARC-1 according to the present invention, as well as for the identification of active molecules, such as those described to continuation. Such cells can be used as a system of model to study hypercholesterolemia, more specifically the ADH and / or disorders of lipid metabolism and lipoproteins These cells can be maintained in a medium of suitable culture, such as DMEM, RPMI, HAM, etc., in any appropriate culture device (plate, flask, disc, tube, bag, etc.)

Diagnosis

The invention then provides diagnostic procedures based on a control of the alteration in the locus of the PCSK9 gene in a subject. Within the context of the present invention, the term "diagnosis" includes detection, control, dosage, comparison, etc., in several stages, including early, presymptomatic and late stages, in adults, children and before of birth.

The diagnosis as a rule includes the prognosis, evaluation of a predisposition or risk of development, the characterization of a subject to define the most appropriate treatment (drug-genetic), etc.

The present invention discloses a method of detecting the presence of hypercholesterolemia or its predisposition, more specifically ADH, and / or disorders of lipid and lipoprotein metabolism in a subject, the method comprising (i) provide a sample of the subject and (ii) detect the presence of an alteration in the locus of the PCSK9 gene in said sample, the presence of said alteration is indicative of the presence of hypercholesterolemia or predisposition to it, more specifically of ADH and / o of disorders of lipid and lipoprotein metabolism. Preferably, said alteration is a nucleotide substitution. More preferably, the invention relates to a method of detecting the presence of ADH or the predisposition thereto.

The present invention discloses a method of detecting the presence of hypercholesterolemia or its predisposition, more specifically of ADH or disorders of lipid and lipoprotein metabolism in a subject, the method comprising detecting in a sample of a subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, the presence of said alteration of the presence or predisposition to hypercholesterolemia, more specifically ADH or lipid and lipoprotein metabolism disorders being indicative. In a more preferred embodiment, said alteration reduces, modifies or eliminates the activity of NARC-1. The process is preferably carried out in vitro or ex vivo .

The present invention relates to procedure defined in claims 1 and 2.

In an embodiment of the present invention, a sample of a subject is extracted non-invasively.  The sample can be a body fluid or a tissue. For example, The sample can be selected from saliva, urine, feces, mucus, secretions, sweat, tears, milk, semen, flow vaginal and vaginal bleeding or body fluids. The sample it can also be a biological tissue or exudate selected from between tonsils, nasal passages, oral tissue, tissue of the inciting membrane, squamous cells of the skin, nails, hair and hair roots

In another embodiment of the present Invention, the sample is from blood.

The present invention discloses a procedure for detecting the presence or predisposition to hypercholesterolemia, more specifically ADH and / or disorders of the lipid and lipoprotein metabolism in a subject, comprising the procedure the detection in a sample of the subject of the presence of an alteration in the mRNA of NARC-1 in said sample, the presence of said alteration is indicative of the presence of hypercholesterolemia or predisposition to it, more specifically of ADH, and / or metabolic disorders of lipids and lipoproteins. Preferably, said alteration is a nucleotide substitution. More preferably, the invention is refers to a procedure for detecting the presence of ADH or the predisposition to it. Optionally, said procedure It comprises a previous stage of contribution of a sample of the subject.

The present invention discloses a procedure for detecting the presence or predisposition to hypercholesterolemia, more specifically ADH and / or disorders of the lipid and lipoprotein metabolism in a subject, comprising the procedure the detection in a sample of the subject of the presence of an alteration in the polypeptide NARC-1 in said sample, the presence of said alteration is indicative of the presence of hypercholesterolemia or of predisposition to it, more specifically of ADH, and / or of disorders of lipid and lipoprotein metabolism. Preferably, said alteration is an amino acid substitution. More preferably, the invention relates to a process of detection of the presence of ADH or predisposition to it. Optionally, said procedure comprises a previous stage of contribution of a sample of the subject.

The present invention discloses a method of evaluating the response of a subject to a treatment of hypercholesterolemia, more specifically ADH, and / or disorders of lipid and lipoprotein metabolism, the method comprising detection in a sample of the subject of the presence of an alteration in the locus of the PCSK9 gene, in the NARC-1 mRNA or in the NARC-1 polypeptide in said sample, the presence of said alteration is indicative of a specific response to said treatment. Preferably said alteration is a substitution of nucleotides or amino acids. More preferably, the invention describes a method of evaluating the response of a subject to an ADH treatment. Optionally, said procedure comprises a previous step of providing a sample of the subject.

The present invention discloses a method of detecting the presence of hypercholesterolemia or its predisposition, more specifically of ADH, and / or disorders of lipid and lipoprotein metabolism in a subject, the method comprising detection in a sample of the subject of the presence of an alteration in the PCSK9 gene, in the LDL receptor gene and / or in the apolipoprotein B gene in said sample, the presence of said alteration is indicative of hypercholesterolemia or predisposition to same, more specifically ADH, and / or lipid and lipoprotein metabolism disorders. Also, the alteration can also be detected at the protein level. More preferably, the invention describes a method of detecting the presence of ADH or its predisposition. Optionally, said procedure comprises a previous step of providing a sample of the subject.

An alteration in the gene can be any form of mutation or mutations, of elimination or deletions, of reconfiguration or reconfigurations and / or insertions in the zone of coding and / or non-coding of the locus, alone or in several combinations Mutations more specifically include point mutations, such as those described above. In a preferred embodiment of the present invention, the alteration is a replacement of nucleotides or amino acids.

Detection of the presence of an altered PCSK9 gene or an altered NARC-1 mRNA sequence according to the present invention can be carried out by sequencing, for example, all or part of the PCSK9 polypeptide or RNA gene, by selective hybridization or by selective enlargement

A more specific embodiment comprises the detection of the presence of a polymorphism as disclosed in Table 2 in the sequence of the PCSK9 gene or in the NARC-1 mRNA of a subject.

More specifically, the alteration of the locus of the PCSK9 gene is detected by segregating a haplotype with the mutation that produces ADH, more preferably the haplotype (polymorphisms B (absence or insertion), H, I, M and U of Table 2).

Preferably, the alteration detected in the locus of the PCSK9 gene or in the NARC-1 mRNA is selected from the group consisting of a replacement of nucleotide T at position 625 and 890 of the SEC. ID. No: 1 and a combination thereof, more preferably a T → substitution at position 625 of the SEC. ID. No. 1, a substitution T → C at position 890 of the SEC. ID. nº: 1 and a combination thereof.

Alternatively, the alteration detected in the locus of the PCSK9 gene or in the NARC-1 mRNA is selected from the group consisting of a substitution of nucleotide A at position 898 and a substitution of nucleotide C at position 953 of SEQ. ID. No. 1 and a combination thereof, more preferably an A → T substitution at position 898 of the SEC. ID. No. 1, a substitution C → T at position 953 of the SEC. ID. nº: 1 and a combination thereof.

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Preferably, the alteration detected in the NARC-1 protein is selected from the group constituted by a replacement of the rest of serine in the position 127 of SEC. ID. nº: 2, a substitution of the rest of phenylalanine in position 216 of the SEC. ID. nº: 2 and a combination of themselves, more preferably a substitution of the serine moiety in SEC position 127. ID. No .: 2 for an arginine (S127R) or a replacement of the phenylalanine residue at position 216 of the SEC. ID. nº: 2 for a leucine (F216L) or a combination of same.

Alternatively, the alteration detected in the NARC-1 protein can also be selected from the group consisting of a substitution of the arginine residue in the SEC position 218. ID. nº: 2, a substitution of the rest arginine at position 237 of the SEC. ID. nº: 2 and a combination thereof, more preferably a substitution of the rest arginine at position 218 of the SEC. ID. nº: 2 for a serine (R218S) or a substitution of the rest of arginine at position 237 of the SEC. ID. No .: 2 for a tryptophan (R237W) or a combination of the same.

The present invention describes a process for detecting the presence of hypercholesterolemia or a predisposition to it, more specifically ADH and / or disorders of lipid and lipoprotein metabolism in a subject, comprising the procedure (i) provide a sample of the subjects and (ii) detect the presence of an RNA of Altered NARC-1 and / or polypeptide expression, the presence of said altered NARC-1 RNA and / or the polypeptide expression is indicative of the presence or predisposition to hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism. More preferably, the invention discloses a method of detection of the presence of ADH or predisposition to ADH.

The present invention describes a method of evaluating the response of a subject to a treatment of hypercholesterolemia, more specifically ADH, and / or disorders of lipid and lipoprotein metabolism in a subject, the method comprising (i) providing a sample of the subject and (ii) detecting the presence of an altered NARC-1 RNA and / or expression of the polypeptide, the presence of said altered NARC-1 RNA and / or expression of the polypeptide is indicative of a specific response to said treatment. More preferably, the invention discloses a method of detecting the presence of ADH or the predisposition to
same.

Altered RNA expression includes the presence of an RNA sequence, altered the presence of a cutting and splicing or elaboration of altered RNA, the presence of a altered amount of RNA, etc.

These can be detected by various techniques known in the art, including sequencing of all or part of NARC-1 RNA or by selective hybridization or selective expansion of all or part of said RNA, for example.

Polypeptide expression Altered NARC-1 includes the presence of a altered polypeptide sequence, the presence of an amount altered NARC-1 polypeptide, the presence of a altered tissue distribution, etc. These can be detected by several techniques known in the art, including sequencing and / or binding to specific ligands (such as antibodies), by example.

The present invention describes a process for detecting the presence of hypercholesterolemia or a predisposition to it, more specifically ADH and / or disorders of lipid and lipoprotein metabolism in a subject, comprising the procedure (i) provide a sample of the subject and (ii) detect the presence of an activity Altered NARC-1, the presence of said RNA from Altered NARC-1 and / or polypeptide expression is indicator of the presence or predisposition to hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism. Preferably, said altered NARC-1 activity It is a diminished NARC-1 activity. Plus preferably, the invention discloses a method of detection of the presence or predisposition to ADH.

The present invention describes a process of evaluating the response of a subject to a treatment of hypercholesterolemia, more specifically of ADH, and / or of the disorders of lipid and lipoprotein metabolism in a subject, comprising the procedure (i) provide a sample of the subject and (ii) detect the presence of an activity of Altered NARC-1, the presence of said activity of Altered NARC-1 is indicative of a response specific to said treatment. Preferably, said activity of Altered NARC-1 is a decreased activity of NARC-1 More preferably, the invention gives know a procedure for detecting the presence of ADH or the predisposition to it.

The present invention discloses a genotyping method in at least one polymorphism of the PCSK9 gene, preferably included in Table 2, which comprises (i) providing a sample of the subject and (ii) determining the identity of the allele of said polymorphism in Said sample. The identity of the allele is preferably determined by carrying out a hybridization test, a sequencing test, a microsequencing test and a specific allele extension test.

The present invention relates to a genotyping method defined in claim 8.

The present invention also describes a method of determining the existence of an association between a polymorphism and a disease or disorder, comprising the following steps: (i) genotyping of at least one PCSK9 gene polymorphism, preferably the one listed in the Table 2, in a population suffering from said disease or disorder; (ii) genotyping of said polymorphism in a reference population; and, (iii) determine if there is a statistically significant association between said disease or disorder and said polymorphism.

As indicated above, various techniques known in the art can be used to detect or quantify the expression or sequence of the altered PCSK9 gene or RNA, including sequencing, hybridization, amplification and / or binding to specific ligands (such as antibodies). Other suitable procedures include the allele-specific oligonucleotide (ASO), the allele-specific extension, the Southern blot (for the DNA), the Northern blot (for the RNAs), the single-chain configuration analysis (SSCA), PFGE , fluorescent in situ hybridization (FISH), gel migration, gel electrophoresis with assured denaturation, heterodual analysis, RNAse protection, chemical incompatibility excision, ELISA, radioimmunoassay (RIA) and immunoenzymatic analysis (IEMA).

Some of these methods (e.g., SSCA and CGGE) they are based on a change in the electrophoretic mobility of acids nucleic, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is observed by a mobility displacement in the gels. The fragments can sequenced next to confirm the alteration.

Some others are based on specific hybridization between the subject's nucleic acids and a specific probe for the natural or altered PCSK9 gene or RNA. The probe may be suspended or immobilized on a substrate. The probe is marked by general rule to facilitate the detection of hybrids. By "specific hybridization" is meant a hybridization under severe conditions.

Some of these methods are particularly suitable for evaluating a polypeptide sequence or the level of expression, such as Northern blotting, ELISA and RIA. These The latter require the use of a specific ligand for the polypeptide, more preferably of a specific antibody.

Sequencing can be carried out using techniques well known in the art, which use automatic sequencers. Sequencing can be carried out in the complete PCSK9 gene or, more preferably, in the specific domains thereof, as a rule those known or capable of carrying harmful mutations or other alterations.

The extension can be done according to several techniques known in the art, such as by reaction in polymerase chain (PCR), ligase chain reaction (CSF), chain shift extension (SDA) and extension based on the nucleic acid sequence (NASBA). These techniques can be carried out using reagents and protocols available in the market. Preferred techniques use PCR specific for the allele or PCR-SSCP. Enlargement usually, to start the reaction, the use of specific primers for nucleic acid.

In this regard, the present invention describes a nucleic acid primer useful for extending the sequences of the PCSK9 gene or locus. Said primers are preferably complementary to nucleic acid sequences in the locus of the PCSK9 gene and specifically hybridize under severe conditions thereto. Specific primers can hybridize specifically under severe conditions with a part of the locus of the PCSK9 gene flanking a target area of said locus, said zone comprising an alteration according to the present invention, more specifically a replacement of nucleotide T at position 625 and / or 890 of SEC. ID. nº: 1 or a polymorphism listed in Table 2, said target area being preferably altered in certain subjects suffering from ADH.

The present invention describes a pair of nucleic acid primers, wherein said pair comprises transcribed and complementary primers, and in which said transcribed and complementary primers specifically extend a PCSK9 gene or an RNA or a target zone thereof, said zone comprising an alteration according to the present invention, more specifically a replacement of nucleotide T at position 625 and / or 890 of the SEC. ID. nº: 1 or a polymorphism included in Table 2, said target area being altered in certain subjects suffering from hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism.

In a more specific embodiment, the invention discloses a nucleic acid primer, wherein said primer is complementary to a part of the coding sequence of PCSK9 and specifically hybridizes under severe conditions with a part thereof ( eg, gene or RNA), wherein said part comprises an alteration according to the present invention, more specifically a replacement of nucleotide T at position 625 and / or 890 of the SEC. ID. nº: 1 or a polymorphism included in Table 2. Preferably, said alteration is present in certain subjects suffering from hypercholesterolemia, more specifically ADH and / or disorders of lipid and lipoprotein metabolism. In this regard, the specific primers of the present invention are specific for the altered sequences in a PCSK9 or RNA gene. Using said primers, the detection of an enlargement product indicates the presence of an alteration in the locus of the PCSK9 gene. In contrast, the absence of the enlargement product indicates that the specific alteration is not present in the sample. More preferably, said primers comprise the nucleotide at position 625 and / or 890 of the SEC. ID. No. 1 or nucleotide at position 5158 and / or 13539 of the SEC. ID. nº: 3. Alternatively, said primers comprise a polymorphism listed in the
Table 2.

Typical primers of the present invention are single stranded nucleic acid molecules of about 5 to 60 nucleotides in length, more preferably about 8 to about 25 nucleotides in length. The sequence can come directly from the sequence of the locus of the PCSK9 gene. Perfect complementarity is preferred, to ensure high specificity. However, certain incompatibilities can be tolerated.

A specific detection technique involves the use of a specific or altered PCSK9 or RNA specific nucleic acid probe, followed by the detection of the presence of a hybrid. The probe may be suspended or immobilized on a substrate or support (such as in a nucleic acid matrix or chip technologies). The probe, as a rule, is marked to facilitate the detection of hybrids.

In this regard, a specific embodiment of the present invention comprises contacting the subject's sample with a specific nucleic acid probe for an altered locus of the PCSK9 gene and assessing the formation of a hybrid. In a specific preferred embodiment, the method comprises simultaneously contacting the sample with a series of probes that are specific, respectively for the locus of the natural PCSK9 gene and for several altered forms thereof. In this embodiment, it is possible to directly detect the presence of various forms or alterations in the locus of the PCSK9 gene in the sample. Likewise, several samples from several subjects can be treated in parallel.

The present invention discloses a nucleic acid probe specific for a PCSK9 or RNA gene. Within the context of the present invention, a probe refers to a polynucleotide sequence that is complementary to (the target portion of a) PCSK9 or RNA gene and is capable of specific hybridization under severe conditions therein and that is suitable for detect polynucleotide polymorphisms, preferably the polymorphism associated with PCSK9 alleles that predispose to or are associated with ADH. The probes are preferably perfectly complementary to the PCSK9 gene, RNA, or target portion thereof. Probes generally comprise single stranded nucleic acids between 8 to 1,000 nucleotides in length, for example between 10 and 800, more preferably between 15 and 700, typically between 20 and 500. It should be understood that longer probes can also be used . A preferred probe of the present invention is a single stranded nucleic acid molecule between 8 to 500 nucleotides in length, which can specifically hybridize under severe conditions with a zone of a PCSK9 gene or the RNA that carries out an alteration.

A preferred embodiment of the present invention is a specific nucleic acid probe for an altered PCSK9 gene or RNA (eg, mutated), that is, a nucleic acid probe that specifically hybridizes under severe conditions to said PCSK9 gene or altered RNA and essentially does not hybridize under severe conditions to a PCSK9 or RNA gene that lack such alteration. Specificity indicates that hybridization to the target sequence generates a specific signal that can be distinguished from the signal generated by non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to the present invention. It should be understood, however, that certain incompatibility can be tolerated, provided that the specific signal can be distinguished from non-specific hybridization.

Specific examples of such probes are nucleic acid sequences complementary to a target portion of the PCSK9 or RNA gene that carries the nucleotide at position 625 and / or 890 of the SEC. ID. No. 1, the nucleotide at position 5158 and / or 13539 of the SEC. ID. No. 3, a polymorphism listed in Table 2 or a mutation described in Table 4.

The sequence of the probes can be derived from the sequences of the PCSK9 and RNA gene provided in the present application. Nucleotide substitutions can be made, as well as chemical modifications of the probe. Such chemical modifications can be carried out to increase the stability of the hybrids (eg interleaver groups) or to label the probe. Typical examples of markers include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labeling, etc.

As indicated above, the alteration in the locus of the PCSK9 gene can also be detected by identifying the alteration or alterations in the NARC-1 polypeptide sequence or expression levels. In this regard, a specific embodiment of the present invention comprises contacting the sample with a specific ligand of an altered NARC-1 polypeptide and determining the formation of a complex.

Different types of ligands can be used, such as specific antibodies. In one embodiment specific, the sample is contacted with an antibody specific for an altered NARC-1 polypeptide and The formation of an immune complex is determined. Various procedures to detect an immune complex can used, such as ELISA, radioimmunoassay (RIA) and assays immunoenzymatic (IEMA).

In a specific embodiment, the procedure comprises contacting a sample of the subject with (a support coated with) an antibody specific for a form of an altered NARC-1 polypeptide and Determine the presence of an immune complex. In a form of specific embodiment, the sample can be contacted simultaneously, in parallel or successively, with several (supports coated with) specific antibodies for different forms of a NARC-1 polypeptide, such as a natural one and several altered forms of it.

Specific examples of such ligands specific are the antibodies specific for the sequence of the altered NARC-1 polypeptide from any mutation at position 127 and / or 216, more particularly a replacement of the serine residue at position 127 with an arginine (S127R) or a substitution of the phenylalanine residue in the position 216 for a leucine (F216L) or any combination of these mutations

The invention also describes a diagnostic kit comprising products and reagents for detecting in a sample of a subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, in the NARC-1 RNA or the expression of the polypeptide and / or in the activity of NARC-1. Optionally, said diagnostic kit further comprises reagents for detecting in a sample of a subject the presence of an alteration in the LDI, receptor and / or apolipoprotein B. Said diagnostic kit according to the present invention comprises any primer, any pair of primers , any nucleic acid probe and / or any antibody described in the present invention.

Said diagnostic kit according to the present invention may further comprise reagents and / or protocols for perform a hybridization, enlargement or immune reaction antigen-antibody.

The present invention relates to a kit of diagnosis defined in claims 9 and 10.

ID

The present invention also provides new targets and method for the identification of experimental or first-line drugs. Such experimental or first-line drugs are useful for developing a treatment against hypercholesterolemia, more particularly ADH, disorders of lipid and lipoprotein metabolism, atherosclerosis and / or CVD. Preferably, said experimental or first-line drugs are useful for developing a treatment against ADH. The procedures include fixation assays and / or functional assays and can be performed in vitro , in cellular systems, in animals, etc. Functional tests comprise, but are not limited to, the cleavage of a substrate. In vitro assays , cell-based assays and animal-based assays involve an NARC-1 protein, preferably an NARC-1 protein comprising an alteration according to the present invention. Optionally, said assays comprise a control with a natural NARC-1 protein.

For cellular systems, cells can be natural, that is, cells that normally express the NARC-1 polypeptide, such as biopsy or expanded in the cell culture. Preferably, these natural cells proceed of the liver or small intestine. Alternatively, the cells they are recombinant host cells that express NARC-1, more specifically to protein NARC-1 comprising an alteration according to the present invention

The invention discloses procedures for the protein target protein identification NARC-1, preferably a protein NARC-1 comprising an alteration according to the present invention

The invention describes methods for the identification of compounds that modulate the activity of NARC-1 Such compounds, for example, can increase or decrease the affinity and / or the rate of fixation of the NARC-1 protein to the substrate, compete with the substrate for binding to the NARC-1 protein or displace the substrate bound to the NARC-1 protein. Preferably, the invention describes methods for the identification of compounds that increase or restore Natural NARC-1 activity. By activity of NARC-1 "natural" means the activity of the natural NARC-1 protein. In addition, the invention is refers to the procedures for the identification of compounds that inhibit the activity of the altered NARC-1 that comprises an alteration that changes the specificity of the substrate and, so it generates new substrates. Such compounds may block the activity of the altered NARC-1 for New substrate

Therefore, the present invention discloses a method of selecting biologically active compounds, said method comprising contacting a test compound with an altered PCSK9 gene or an altered NARC-1 protein or a fragment thereof. minus 15 consecutive residues comprising an alteration, in which the alteration reduces, modifies or suppresses the activity of NARC-1, and determines the ability of said test compound to modulate the expression and / or activity of said gene, protein or fragment .

The present invention relates to procedure defined in claim 4.

The present invention describes a method of selecting biologically active compounds, said method comprising in vitro contacting a test compound with a PCSK9 gene or an NARC-1 polypeptide, preferably a PCSK9 gene or an NARC-1 polypeptide, or a fragment thereof of at least 15 consecutive residues, comprising an alteration according to the present invention and determining the ability of said test compound to bind said PCSK9 gene or NARC-1 polypeptide. Binding to said gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus affect a series of reactions that lead to hypercholesterolemia, more specifically to ADH, and disorders. of lipid and lipoprotein metabolism in a subject. In a preferred embodiment, the method comprises contacting in vitro a test compound with a NARC-1 polypeptide or a fragment thereof, preferably an NARC-1 polypeptide or a fragment thereof comprising an alteration according to the present invention. , and determining the ability of said test compound to bind to said NARC-1 polypeptide or fragment. The fragment preferably comprises a point of attachment to the substrate of the polypeptide
NARC-1

The present invention describes a method of selecting active compounds against hypercholesterolemia, more particularly ADH and / or disorders of lipid and lipoprotein metabolism, said method comprising in vitro contacting a test compound with a NARC-1 polypeptide or a fragment thereof of at least 15 consecutive residues, preferably an NARC-1 polypeptide or a fragment thereof comprising an alteration according to the present invention, and determining the ability of said test compound to bind to said NARC-1 polypeptide or fragment thereof. The NARC-1 polypeptide or a fragment thereof can be used essentially in pure form, in suspension or immobilized on a support.

In another specific embodiment, the method comprises contacting a host cell recombinant expressing the NARC-1 polypeptide, preferably an NARC-1 polypeptide comprising an alteration according to the present invention, with a compound of test and determine the ability of said test compound to bind said NARC-1 polypeptide and / or modulate the NARC-1 polypeptide activity.

The determination of the fixation can lead to carried out by various techniques, such as by marking the compound of trial, by competition with a labeled reference ligand, Identification analysis of two hybrids, etc. Modulation of the activity includes, without limitation, the inhibition or activation of the autocatalytic elaboration of pro-NARC-1 and / or inhibition or activation of substrate cleavage, more specifically a synthetic substrate comprising a processing area of zymogen

The present invention discloses a method of selecting biologically active compounds, said method comprising in vitro contacting a test compound with an NARC-1 polypeptide, preferably an NARC-1 polypeptide comprising an alteration according to the present invention, and determining the ability of said test compound to modulate the activity of said NARC-1 polypeptide.

The present invention discloses a method of selecting biologically active compounds, said method comprising in vitro contacting a test compound with a PCSK9 gene, preferably a PCSK9 gene comprising an alteration according to the present invention, and determining the ability to said test compound to modulate the activity of said PCSK9 gene.

The invention also describes methods for selecting biologically active compounds that use non-human transgenic animals that express an NARC-1 protein, preferably an NARC-1 protein comprising an alteration according to the present invention. Optionally said non-human transgenic animals can be homozygous or heterozygous for the altered PCSK9 gene. Said methods comprise (i) administering a test compound to said non-human transgenic animal and (ii) determining the ability of said test compound to modulate the activity of NARC-1. Said activity of NARC-1 can be evaluated by determining the plasma concentration of cholesterol and / or lipoparticles (VLDL, IDL, LDL), determining the plasma enzymatic activity of NARC-1, analyzing some tissues (liver, small intestine), determining the kinetics of lipoproteins. The enzymatic activity of NARC-1 can be determined with the synthetic substrate, as described in Seidah et al . (2003).

The above identification analyzes can be performed on any suitable device, such as plates, tubes, discs, flasks, etc. As a general rule, the analysis is Performs in plates with many wells. Various test compounds They can be analyzed in parallel.

In addition, the test compound may be of Origin, nature and varied composition. Can be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., isolated or mixed with other substances. The compounds can be, by example, all or part of a combinatorial product bank. The Test compounds may be a complementary RNA or an RNAi. Test compounds may be competitive or substrates suicidal "Suicidal substrate" means a compound that, after joining the NARC-1 protein, the group reagent forms an irreversible bond with NARC-1 inactivating it.

       \ vskip1.000000 \ baselineskip
    

Therapy

The invention discloses methods of hypercholesterolemia treatment, more particularly ADH, disorders of lipid and lipoprotein metabolism, atherosclerosis and / or CVD. Preferably, the invention describes Hypercholesterolemia treatment procedures, more particularly ADH and / or lipid metabolism disorders and lipoproteins due to a protein alteration NARC-1

The invention also describes a process. of treatment or prevention of hypercholesterolemia, more specifically of ADH, lipid metabolism disorders and lipoproteins, atherosclerosis and / or CVD in a subject, comprising the procedure the administration to said subject of a polypeptide Functional NARC-1 (e.g., natural) or an acid nucleic encoding it. More preferably, the invention gives to know a procedure of treatment or prevention of ADH.

The invention describes the use of a NARC-1 or a nucleic acid functional polypeptide encoding it, in the preparation of a composition Pharmaceutical for the treatment or prevention of hypercholesterolemia, more specifically of ADH, disorders of the lipid and lipoprotein metabolism, atherosclerosis and / or CVD in a subject. More preferably, the invention describes a pharmaceutical composition for the treatment or prevention of ADH.

The invention also describes a process. of treatment or prevention of hypercholesterolemia, more particularly of ADH, lipid metabolism disorders and lipoproteins, atherosclerosis and / or CVD in a subject, comprising the procedure the administration to said subject of a compound which modulates the expression and / or activity of NARC-1. More preferably, the invention describes a method of treatment or prevention of ADH. The invention discloses furthermore a pharmaceutical composition comprising a compound that modulates the expression and / or activity of NARC-1.

The invention generally discloses, the use of a compound that modulates expression and / or activity  of NARC-1 in the preparation of a composition Pharmaceutical for the treatment or prevention of hypercholesterolemia, more particularly ADH, disorders of the lipid and lipoprotein metabolism, atherosclerosis and / or CVD in a subject. More preferably, the invention describes a pharmaceutical composition for the treatment or prevention of ADH.

The present invention demonstrates the etiological link between hypercholesterolemia, more particularly ADH and an alteration of the locus of the PCSK9 gene. The invention therefore provides a new therapeutic intervention target. Several methods can be contemplated to restore or modulate the activity or function of NARC-1, more specifically the activity or function of normal NARC-1, in a subject, particularly those carrying an altered locus of the PCSK9 gene. The supply of natural function to said subject is expected to suppress the phenotypic expression of hypercholesterolemia, more specifically of ADH, in a cell or pathological organism. The contribution of this function can be done by gene or protein therapy or by administering compounds that modulate the activity of NARC-1.

If protein alteration NARC-1 leads to a decrease or loss of NARC-1 activity, the treatment consists of administer a biologically active compound that increases or resets the activity of NARC-1. Said compound biologically active can be a NARC-1 protein natural. Alternatively, said compound may be an activator. of the NARC-1 protein. Said compound may also increase protein expression NARC-1

If the alteration of the NARC-1 protein leads to a new specificity of a substrate, the treatment consists in administering a biologically active compound that inhibits the activity of the altered NARC-1 protein. Said compound may decrease the expression of the NARC-1 protein. For example, said compounds may be a complementary RNA or an siRNA of the PCSK9 gene comprising the alteration that gives rise to ADH. Alternatively, said compound may be an altered NARC-1 protein inhibitor. Said compound may compete with the substrate or it may be a suicide substrate.

Other aspects and advantages of the present invention in the following experimental section, which should be considered illustrative and not limiting the scope of the present invention

Examples

The inventors mapped a third HCHOLA3 locus in 1p32 and then describe two mutations in the PCSK9 gene that produces ADH. PCSK9 encodes NARC-1 (convertase regulated by neuronal apoptosis). Its mutations lead to reduced activation of the enzyme. The kinetics of lipoprotein in the probands revealed an overproduction of the particles rich in apoB100 which shows that the pathogenic origin of the disease is hepatic. In conclusion, NARC-1 is a newly identified human subthylase that contributes to cholesterol homeostasis and is the first example of a dominant disease associated with a defect in a member of the large subtylase family.

To identify the HCHOLA3 locus (formerly FH3), which the inventors mapped (Varret et al ., 1999) to 1p34.1-p32 (OMIM603776) and was confirmed by Hunt et al . In a large family in Utah (Hunt et al ., 2000), the inventors carried out position cloning using the originally connected family and 23 French families in which the involvement of the LDLR and APOB genes had been excluded.

The HC92 family was identified by testing (HC92-II-7) that belongs to a multiple ADH family tree from which samples of twenty-nine family members were taken and analyzed in parametric connection analysis. In the reduced family tree studied in the connection analysis, 12 subjects had total cholesterol concentrations higher than 97.5 percentile when compared with other French individuals of equal age and sex (Steinmetz, 1990) (total average cholesterol: 3.63 g / l ± 0.68, LDL-medium cholesterol: 2.87 ± 0.72 g / l). The inventors excluded the connection to the LDLR and APOB genes [lod scores at -14.05 and -10.01 (? = 0.0), respectively]. The family was genotyped by 8 Genethon markers in the 1p34-p32 zone (Fig. 1). The inventors obtained very significant lod scores with a maximum of 4.26 (? = 0.0) at D1S2742 which reached 4.80 in the multipoint analyzes (Table 1, Fig. 3a). The haplotype analysis identified a critical interval of 5.9 Mb between D1S231 and D1S2890 . The critical range that the inventors team had previously described in the HC2 family (Varret et al ., 1999) was between markers D1S472 and D1S211 , therefore more distal. The re-examination of the haplotype data (Fig. 2) showed that all affected subjects of the HC2 family also shared the same haplotype between markers D1S2722 and D1S2890 except HC2-II-5. This "affected" subject presented a case of recombination in D1S211 thus providing the centromeric boundary of the area described in 1999. Consequently, all family members were investigated again. HC2-II-5 (who refuses treatment) was the only subject that presented a significant variation (a marked elevation of triglycerides) and therefore no longer adapts to the inclusion criteria.

The inventors created the physical mapping of the experimental zone between D1S197 and D1S2890 comprised of 82 overlapping BAC sequences extracted from the Human Genome Project. The zone between D1S197 and D1S2890 contains 41 genes among which 8 encode interesting functional experimental compounds with respect to lipid metabolism: EPS 15 (substrate-15 of the series of epidermal growth factor receptor reactions), OSBPL9 (pseudoprotein 9 that binds to oxisterol), SCP2 (sterol carrier protein 2), LRP8 (protein 8 related to low density lipoprotein receptor), DHCR24 (24-dehydrocholesterol reductase), PRKAA2 (protein kinase, activated by AMP, catalytic subunit) alpha 2), DAB1 (counterpart 1 deactivated) and PCSK9 (encoding NARC-1). This convertase 1 regulated by neuronal apoptosis is a supposed new proprotein convertase (PC) that belongs to the subfamily subfamily (Seidah et al ., 2003). A related protein is subtilisin kexin isoenzyme-1 (SKI-1) / zone-1-protease (S1P) known because it plays a key role in cholesterol homeostasis by making proteins that bind to the sterol regulatory element ( SREBP) (Brown and Goldstein, 1999; Elagoz et al ., 2002). The cDNA comprises 3617 base pairs that encode a 692 amino acid protein. NARC-1 was mapped for 1p33-p34.3. The inventors accurately located their cDNA using the Blast program (http://www.ncbi.nlm.nih.gov/BLAST/) in the HCHOLA3 interval as follows: tel D1S231-D1S2661-D1S417-D1S2652-PCSK9-D1S475 -D1S200-D1S2742-cen.

The systematic bidirectional sequencing of the 125 exons of the first seven candidates showed no mutation in the probands. By sequencing the 12 exons of PCSK9, the inventors identified in the HC92 family a T → substitution in exon 2 in nucleotide 625 that provides for a substitution in codon 127 of arginine by the conserved serine (S127R), thereby creating a zone of cleavage Mnl l (Fig. 3b, Fig. 4). The substitution was analyzed in the HC92 family members and 100 references. It was absent in 200 reference chromosomes indicating that it is not a polymorphism. It was discovered in 12 affected family members and in a subject HC92-IV-3 that has a total cholesterol concentration in the 90th percentile when compared to other French individuals of equal age and sex. Therefore, family penetration is estimated at 0.94. Interestingly, the S127R mutation was also found in the HC2 test and was segregated together with the disease in the family except in the HC2-II-5 subject, confirming that it had been poorly classified in the connection analyzes described above ( Varret et al ., 1999). To assess the possible recurrence of this mutation, the inventors tested 5 intragenic polymorphic markers that the inventors had identified in PCSK9 (4 SNPs and a repeat of GCT) in both families. The same haplotype segregated with the S 127 R mutation in both HC2 and HC92 families: (polymorphisms B (no insertion), H, I, M and U) (Tables 2 and 3). In addition, a single haplotype was also obtained for the extragenic markers surrounding PCSK9 ( D1S2661, D1S417, D1S475, D1S200 and D1S2742 ) in both families. These results show that despite the absence of records and different geographical origins, families share a common ancestor. The possibility of a French founder effect was stipulated since the mutation was not found in 22 other French ADH probands.

By systematic bidirectional sequencing of the 12 exons of the PCSK9 gene in 22 ADH probabilities, a second mutation (F216L) was identified in the HC60 family test (Fig. 2c) that died of myocardial infarction at 49 years (Fig. 3 d , Fig. 4). This mutation was segregated with the ADH phenotype in the family and was not found in 200 reference chromosomes. No major reconfiguration was found in any of the Southern transfer probands (data not shown). Therefore, mutations in PCSK9 have been found in 12.5% of the ADH families analyzed.

The inventors also identified 25 polymorphisms present in different probands and in reference chromosomes of subjects with normal cholesterol concentrations (Table 2). These variations and their respective frequencies in the French population are included in Table 2. It should be noted that none of these polymorphisms gives rise to new splicing areas of the donor or recipient (score calculated according to Senapathy et al ., 2003) ( Senapathy et al ., 1990; Shapiro and Senapathy, 1987).

In order to unravel at the molecular level the consequences of the S127R and F216L mutations, the inventors introduced them into the human PCSK9 cDNA (Seidah et al ., 2003). The inventors also obtained four other mutants, namely S127A, S127P, 15_16insL (polymorphic variant in which one more leucine is added in the hydrophobic elongation of the signal peptide) and the H226A mutant of the active zone (Seidah et al ., 2003) . The cDNAs encoding natural NARC-1 (WT) and its mutants contained a V5 epitope with C-terminal, were transfected temporarily into HEK293 cells. A 4 h pulse with Met and Cys labeled with 35 S was followed by immunoprecipitation of cell lysates and the medium with a V5 mAb (Seidah et al ., 2003). The inventors have previously shown that proNARC-1 is synthesized as a 72 kDa precursor that experiences two cases of zymogen cleavage. The first is fast and takes place in the endoplasmic reticulum (ER) in the sequence YVVVL_ {82} \ downarrow, which gives rise to product N1 from 63 to 65 kDa and the prosegment (pro) of 14 kDa. The second is produced with much lower efficiency in the assumed PHVDY_ {142} \ downarrow sequence and gives rise to the supposedly active 58 kDa N2 enzyme (Brown and Goldstein, 1999). Through STORM quantification, the inventors estimate that both the S127R and F216L mutation lead to 3 times lower levels of N2. In addition, the secreted level of N1 was also 2 times lower for the S127R mutant. It is interesting, although the S127A mutant exhibits similar behavior, the S127P resembles WT. Finally, the 15_16insL allele variants seem to give rise to a \2 times higher percentage of the N1 and N2 products, suggesting that more active NARC-1 is produced.

The inventors have identified a new gene involved in ADH by cloning a position. Link analysis was performed in two large French genealogical families: HC92 and HC2 in which the involvement of the LDLR and APOB genes had been excluded. A maximum lod score of 4.26 was obtained for D1S2742 in the HC92 family. Haplotype analysis restricted the connection zone to a 5.9 Mb interval between markers D1S231 and D1S2890 at 1p32. The inventors' team had previously described the location of HCHOLA3 in 1p32-p34.1 by link analysis performed in the HC2 family (Varret et al ., 1999). In this family, the critical range was flanked by markers D1S472 and D1S211 and therefore was more distal compared to that identified in the HC92 family. The re-examination of the haplotype data showed that all affected subjects of the HC2 family also shared the same haplotype between markers D1S2722 and D1S2890 except (HC2-II-5). This "affected" subject presented a case of recombination in D1S211 thereby providing the centromere boundary of the area described in 1999. Therefore, it was investigated again (HC2-II-5). The new lipid measurements had the same high cholesterol but also marked elevated triglycerides. This alteration can be explained by the recent knowledge of an important alcohol intake that currently prevents the appropriate evaluation of the subject's status with respect to the family trait. The identification of the S127R mutation of the PCSK9 gene in all other affected members of the HC2 family and its absence in (HC2-II-5) confirmed that it had been misclassified for genetic analysis. The identification of the S127R mutation of the PCSK9 gene in the HC92 family also helped clarify the genetic status of the 8 children who had been sampled but not included in the linkage analyzes. These results are adapted to the conservative method that the inventors had selected (total cholesterol higher than 97.5 percentile compared to the French population of the same sex and age) and this also allowed reduced penetration. This last parameter was confirmed since the S127R mutation of the PCSK9 gene was identified in (HC92-IV-3) who has a total cholesterol concentration in the 90th percentile (compared to other French individuals of the same age and sex) and presented concentrations higher cholesterol compared to the concentrations of their unaffected sisters (2.5 percentile for HC92-IV-1 and 30 percentile for HC92-IV-2). Therefore, penetration can currently be estimated at 0.94 in the family when considering the inclusion criteria that were applied. This characteristic of a mutation of the PCSK9 gene is also found with the mutations of the LDLR gene (Hobbs et al ., 1989; Sass et al ., 1995) and more generally considering the variability of the hypercholesterolemic phenotype (evaluated by conventional clinical and biological criteria) which may be due to the effect of environmental factors or modifying genes.

The haplotype analysis showed that segregated a single haplotype with the S127R mutation in both families HC92 and HC2. Therefore, it can be assumed that despite the absence of records and of different geographical origins, families They share a common ancestor. The possibility of a founder effect French can be stipulated since the mutation was not found in a total of 22 other French probands (data not shown).

NARC-1 is a new convertase recently cloned by two pharmaceutical companies ( NARC-1 , Millenium Pharmaceuticals and LP251 , Eli Lilly). It was first identified by cloning the cDNAs regulated by augmentation following the apoptosis induced by serum deprivation in primary cerebellar neurons. NARC-1 was more accurately characterized recently by Seidah et al . who used preserved short segments of the SKI-1 catalytic subunit as decoys and the Protein Blast program to identify the convertase in a patented database (Seidah et al ., 2003). It is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular elaboration in the endoplasmic reticulum (ER) in the primary cleavage sequence YVVVLKEE 85 indicative of enzyme specificity (Seidah et al ., 2003) of NARC-1. The excision of the prosegment is necessary for the exit of NARC-1 from the ER. The S127R mutation resides between the sequences of the primary and assumed secondary zymogen of proNARC-1, while F216L is located close to the active sequence (H226). Mainly, the S127R mutation creates an RGD zone that may be involved in integrin binding (Ruoslahti, 1996).

Although only the S127R mutant produces the reduction in the secreted level of N1, both the S127R and F216L mutations result in reduced production of the enzymatically active N2. In addition, the VLDL, IDL and LDL apo B100 kinetics carried out in ADH subjects carrying the S127R mutation showed an overproduction in the liver of lipoprotein rich in apo B100. Therefore, the prevalence of the disease shows that NARC-1 is a speed-limiting enzyme involved in homeostasis of cholesterol in the liver. Although most enzymopathies are inherited recessively, predominance is described in some specific highly regulated enzymes or tissue. This is seen in two types of porphyria: AIP (acute intermittent porphyria) (Desnick et al ., 1985) and PCT (late cutaneous porphyria) (Felsher et al ., 1982), which are caused by an insufficiency in porphobilinogen deaminase and uroporphyrinogen decarboxylase, respectively. However, unlike porphyria, PCSK9 gene defects seem very penetrating. NARC-1 belongs to the subtylase family of mammals with 9 members in which only one other member was known to carry a mutation causing the disease: a heterozygosity of the compound in the PC1 gene causes obesity and endocrinopathy due to the production of prohormone altered (MIM 162150). However, the heterozygosity of one of the mutations is imperceptible, thus suggesting a recessive transmission (Jackson et al ., 1997). Although PCs activate a wide variety of proteins, it is noteworthy that none of them were so far linked to a dominant human disease. NARC-1 is therefore unique in this respect and can lead to the discovery of others.

Although the related SKI-1 / S1P convertase plays a key role in the regulation of cholesterol and fatty acid homeostasis through the elaboration of SREBP1 and SREBP2, the exact implication of NARC-1 in cholesterol homeostasis is still under investigation. It is interesting that NARC-1 is expressed mainly in the liver and small intestine which play main functions in the synthesis and regulation of cholesterol (Seidah et al ., 2003). Since apo B100 concentrations are regulated after translation (Bostrom et al ., 1988), it is possible that NARC-1 could inactivate apo B100 and consequently decrease the LDL concentration. In fact, an alleged sequence LIEIGL? 668 of apo B100 is proposed to respect the requirements of the primary and secondary structure of the selectivity of the NARC-1 elaboration (Seidah et al ., 2003). This may therefore explain the described 7070 kDa form of apo B100 that is observed to occur under stressful cellular conditions (Cavallo et al ., 1999).

The crucial role of NARC-1 is highlights the hypercholesterolemia that occurs when the gene is mutated resulting in decreased activation of NARC-1 The identification of substrate (s) of NARC-1 will help clarify the new mechanisms of the disease and constitutes a target (s) for new intervention strategies to limit the elevation of LDL particles and prevent morbidity and mortality of the premature atherosclerosis.

Procedures Family Selection

French hypercholesterolemic families were selected through 8 lipid offices of the National Network for ADH ("Réseau National de Recherche sur les Hypercholestérolémies Familiales"). The probands were determined among the consecutive subjects of the offices. Inclusion criteria for probands were: total cholesterol higher than 97.5 percentile when compared to the French population of the same sex and age (Steinmetz, 1990), LDL cholesterol greater than 1.9 g / l or 1.6 g / l for children, triglycerides below 1.5 g / l, personal or documented family xanthomas and / or cornea arch and early CVD. Lipid measurements were repeated to determine the existence of isolated primary hypercholesterolemia due to elevated LDL. Family history and family trees were investigated. Parental authorization was obtained from all subjects included in this study. The HC2 family had been described above and described long (Varret et al ., 1999). Functional tests showed normal binding, internalization and degradation of LDL particles in the fibroplasts of the probands (HC2-II-9) (Hobbs et al ., 1989). Five other families (HC35, HC60, HC92, HC122 and HC143) were studied representing 26 affected and 26 unaffected subjects. For affected subjects, total mean cholesterol and LDL were 3.27 g / l ± 0.77 and 2.47 ± 0.76 g / l, respectively.

DNA genotyping and analysis

DNA was isolated from whole blood samples as described above (Collod et al ., 1994). All families were tested with polymorphic markers of the LDLR and APOB genes. For LDLR , two intragenic markers ( D19S584 in intron 1 and (TA) n in exon 18) and two adjacent markers ( D19S394 and D19S221 ) were studied . The 5'HVR (TG repeat) and the 3'HVR (VNTR) for the APOB gene and the screening for the described R3500Q mutation (Rabès et al ., 1997) were studied. Genotyping was performed on 1p34-p32 using 11 microsatellites of Genethon mapping ( D1S472, D1S2722, D1S211, D1S197, D1S231, D1S2661; D1S417, D1S475, D1S200, D1S2742, D1S2890 ) as described in 1994. .

Link analysis

Link analysis was carried out parametric with accepted ADH parameters: dominant transmission of the trait, penetration of 0.9 for heterozygotes and a frequency of the disease allele of 1/500. The programs were used MLINK and LINKMAP (Ott, 1991) and the VITESSE program (O'Connell and Weeks, 1995) to perform two-point LOD score analysis and multipoint. The frequencies of the microsatellite allele were calculated among unrelated family members. The link to the assumption of equal recombination rates female to male

Identification and analysis of the experimental gene

Microsatellites were located in the area 1p34-p32 in the Genome Project sequences Human, a physical mapping of the area agreed with the published by UCSC: http://www.genome.ucsc.edu. Repeat programs  Master and Genscan allowed prediction and identification in the Genbank database of experimental genes from position. The Blast program was used (http://www.ncbi.nlm.nih.gov/BLAST/) to locate exactly Experimental genes The intron / exon structure of the 8 functional candidates and the primers were designed with the Mac Vector® software. 137 pairs of primers to approximately 100 base pairs that surround each Exon Limit PCR were carried out with DNA polymerase thermostable from LAROVA Biochemic GmbH (Germany) in GeneAmp® PCR system 9600 (Perkin Elmer). The fluorescent sequencing with version 1.0 of Big Dye Terminator in the GeneAmp® PCR system 9700 (Perkin Elmer), in the conditions supplied by the manufacturer. We analyzed the electrophoregrams using Sequencing Analysis® 3.4 and SeqED®.

PCSK9 analysis

Primers designed to study the 12 exons of NARC-1 and their amplification conditions were available on demand. The main reconfigurations for NARC-1 were investigated by Southern blot as described (Collot et al ., 1994). A rapid detection method of the S127R mutation was developed using PCR amplification followed by Mn / I digestion. After exon 2 enlargement, the 543 base pair PCR product was digested by the Mn / I enzyme. 5U. After electrophoresis in a 2% agarose gel, the 208, 203 and 60 base pair fragments were distinguished in the normal allele, while 208, 143 and 60 base pair fragments appeared in the mutated alleles ( the normal 203 base pair fragment was divided into 143 and 60 base pair fragments and the two 60 base pair fragments were generated comigrated). The segregation analysis of this mutation in families HC2 and HC92 and the analysis of 200 chromosomes of unaffected persons of French descent were both tested by sequencing and by digestion with Mn / I.

Studies of mutants and NARC-1 protein

HEK293 cells were transfected temporarily with pIRES2 recombinant vectors (Seidah et al ., 2003) expressing natural hNARC-1-V5 (WT) or its mutants H226A, F216L, S127R, S127A, S127P and 15_16insL (+ L). The cells were labeled 24 hours later with pulsations with [35 S] Easy Tag Express mixture for 4 h. The cell extracts and the medium were immunoprecipitated with V5 antibody and the precipitates were resolved by SDS-PAGE in 8% glycine gels.

Registration numbers of the genes tested

EPS15 , NM_001981; OSBPL9 , NM_024586; SCP2 , NM_002979; LRP8 , NM_004631; DHCR24 , NM_014762; PRKAA2 , NM_006252; DAB1 , NM_021080); NARC-1 : AX207686 human (gi: 15422368); Mus musculus:
AX207688; Rattus norvegicus AX207690.

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Maugeais , C., Ouguerram , K .; Mahot , P., Krempf , M. & Magot , T., Diabetes Metab. 22, 57-63 ( 1996 ).

Maugeais , C., Ouguerram , K., Krempf , M. & Magot , T. Clin. Chem. Lab. Med. 36, 739-745 ( 1998 ).

Morganroth , J., Levy , RI., McMahon , AE. & Gotto , AM Jr. J. Pediatr. 85, 639-643 ( 1974 ).

O'Connell , JR. & Weeks DE. Nature Genet 11, 402-408 ( 1995 ).

Ott , J. Analysis of human genetic linkage, revised ed. The Johns Hopkins University Press , Baltimore and London ( 1991 ).

Perusse , L. Arteriosclerosis. 9, 308-318 ( 1989 ).

Pont , F., Duvillard , L., Verges , B. & Gambert , P. Arterioscler. Thromb. Vasc Biol. 18, 853-860 ( 1998 ).

Rabès , JP. et al. Hum. Mutat 10: 160-163 ( 1997 ).

Rice , T., Vogler , GP., Laskarzewski , PM., Perry , TS. & Rao , DC. Hum. Biol. 63, 419-439 ( 1991 ).

Ruoslahti , E. Annu. Rev. Cell. Dev. Biol. 12, 697-715 ( 1996 ).

Saint-Jore , B. et al. Eur. J. Hum. Genet 8, 621-630 ( 2000 ).

Sass , C., Giroux , LM., Lussier-Cacan , S., Davignon , J. & Minnich , A. J. Biol. Chem. 270, 25166-71 ( 1995 ).

Seidah , NG. et al. Proc. Natl Acad. Sci. USA. 100, 928-933 ( 2003 ).

Senapathy , P., Shapiro , MB. & Harris , NL. Methods Enzymol. 183, 252-278 ( 1990 ).

Shapiro , MB. & Senapathy , P. Nucleic Acids Res. 15, 7155-7174 ( 1987 ).

Steinmetz , J., Total Cholesterol. In: Siest G, Henny J, Schiele F (eds). Références in biologie clinique. Elsevier , 1990 . Paris, pages 190-209.

Varret , M. et al. Am. J. Hum. Genet 64, 1378-1387 ( 1999 ).

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 1 Area lod ratings obtained in the family HC92

one

TABLE 2 Polymorphisms identified in the PCSK9 gene

2

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 3 Haplotypes of affected subjects of the three families from ADH

3

       \ vskip1.000000 \ baselineskip
    

Marker haplotypes surrounding PCSK9 mutations: the markers are given in physical order from the telomere (left) to the centromere (right). A common haplotype of the disease is secreted with the S127R mutation in both the HC92 and HC2 families.

TABLE 4 Mutations identified in the PCSK9 gene

4

TABLE 4 (Continued)

5

<110> Institut National de la Santé et de the Medical Recherche

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<120> Mutations in the human PCSK9 gene associated with hypercholesterolemia

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<130> B0202WO

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<150> EP 03 291025.9

         \ vskip0.400000 \ baselineskip
      

<151> 2003-04-25

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<150> US 60 / 538,231

         \ vskip0.400000 \ baselineskip
      

<151> 2004-01-23

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<170> PatentIn version 3.1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 3617

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (245) .. (2320)

         \ vskip0.400000 \ baselineskip
      

<223>

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mutation

         \ vskip0.400000 \ baselineskip
      

<222> (625) .. (625)

         \ vskip0.400000 \ baselineskip
      

<223> T-> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mutation

         \ vskip0.400000 \ baselineskip
      

<222> (890) .. (890)

         \ vskip0.400000 \ baselineskip
      

<223> T-> C

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

         \ vskip1.000000 \ baselineskip
      

6

7

8

9

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 692

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> ACT-SITE

         \ vskip0.400000 \ baselineskip
      

<222> (186) .. (186)

         \ vskip0.400000 \ baselineskip
      

<223> Asp

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> ACT-SITE

         \ vskip0.400000 \ baselineskip
      

<222> (226) .. (226)

         \ vskip0.400000 \ baselineskip
      

<223> His

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> ACT-SITE

         \ vskip0.400000 \ baselineskip
      

<222> (317) .. (317)

         \ vskip0.400000 \ baselineskip
      

<223> Asn

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> ACT-SITE

         \ vskip0.400000 \ baselineskip
      

<222> (386) .. (386)

         \ vskip0.400000 \ baselineskip
      

<223> Be

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> ACT-SITE

         \ vskip0.400000 \ baselineskip
      

<222> (186) .. (186)

         \ vskip0.400000 \ baselineskip
      

<223> Asp

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> SITE

         \ vskip0.400000 \ baselineskip
      

<222> (127) .. (127)

         \ vskip0.400000 \ baselineskip
      

<223> replacement Ser -> Arg

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> SITE

         \ vskip0.400000 \ baselineskip
      

<222> (216) .. (216)

         \ vskip0.400000 \ baselineskip
      

<223> Phe replacement -> Leu

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> VARIANTE

         \ vskip0.400000 \ baselineskip
      

<222> (46) .. (46)

         \ vskip0.400000 \ baselineskip
      

<223> Leu

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> VARIANTE

         \ vskip0.400000 \ baselineskip
      

<222> (53) .. (53)

         \ vskip0.400000 \ baselineskip
      

<223> Val

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> VARIANTE

         \ vskip0.400000 \ baselineskip
      

<222> (474) .. (474)

         \ vskip0.400000 \ baselineskip
      

<223> Val

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> VARIANTE

         \ vskip0.400000 \ baselineskip
      

<222> (670) .. (670)

         \ vskip0.400000 \ baselineskip
      

<223> Gly

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

10

eleven

12

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 26220

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (980) .. (1186)

         \ vskip0.400000 \ baselineskip
      

<223> exon 1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (4985) .. (5176)

         \ vskip0.400000 \ baselineskip
      

<223> exon 2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (7665) .. (7788)

         \ vskip0.400000 \ baselineskip
      

<223> exon 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (13417) .. (13550)

         \ vskip0.400000 \ baselineskip
      

<223> exon 4

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (13788) .. (13929)

         \ vskip0.400000 \ baselineskip
      

<223> exon 5

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (17130) .. (17326)

         \ vskip0.400000 \ baselineskip
      

<223> exon 6

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (18464) .. (18647)

         \ vskip0.400000 \ baselineskip
      

<223> exon 7

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (19169) .. (19342)

         \ vskip0.400000 \ baselineskip
      

<223> exon 8

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (19632) .. (19780)

         \ vskip0.400000 \ baselineskip
      

<223> exon 9

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (20605) .. (20782)

         \ vskip0.400000 \ baselineskip
      

<223> exon 10

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (22494) .. (22675)

         \ vskip0.400000 \ baselineskip
      

<223> exon 11

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> exon

         \ vskip0.400000 \ baselineskip
      

<222> (24488) .. (24703)

         \ vskip0.400000 \ baselineskip
      

<223> exon 12

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> 3'UTR

         \ vskip0.400000 \ baselineskip
      

<222> (24488) .. (24703)

         \ vskip0.400000 \ baselineskip
      

<223>

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> 5'UTR

         \ vskip0.400000 \ baselineskip
      

<222> (749) .. (979)

         \ vskip0.400000 \ baselineskip
      

<223>

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mutation

         \ vskip0.400000 \ baselineskip
      

<222> (5158) .. (5158)

         \ vskip0.400000 \ baselineskip
      

<223> T-> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> mutation

         \ vskip0.400000 \ baselineskip
      

<222> (13539) .. (13539)

         \ vskip0.400000 \ baselineskip
      

<223> T \ rightarrowC

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (916) .. (916)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (1022) .. (1042)

         \ vskip0.400000 \ baselineskip
      

<223> CTG with elongation - insertion of leucine

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (1116) .. (1116)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (1120) .. (1120)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (1137) .. (1137)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (4824) .. (4824)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (7464) .. (7464)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13327) .. (13327)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13349) .. (13349)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13406) .. (13406)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13559) .. (13559)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13626) .. (13626)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13632) .. (13632).

         \ vskip0.400000 \ baselineskip
      

<223> G

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13753) .. (13753)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13781) .. (13781)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13932) .. (13932)

         \ vskip0.400000 \ baselineskip
      

<223> G

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (13993) .. (13993)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (19444) .. (19444)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (19576) .. (19576)

         \ vskip0.400000 \ baselineskip
      

<223> C

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (19657) .. (19657)

         \ vskip0.400000 \ baselineskip
      

<223> A

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (19697) .. (19697)

         \ vskip0.400000 \ baselineskip
      

<223> G

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (20845) .. (20845)

         \ vskip0.400000 \ baselineskip
      

<223> T

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (20846) .. (20846)

         \ vskip0.400000 \ baselineskip
      

<223> G

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (22769) .. (22769)

         \ vskip0.400000 \ baselineskip
      

<223> G '

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> allele

         \ vskip0.400000 \ baselineskip
      

<222> (24633) .. (24633)

         \ vskip0.400000 \ baselineskip
      

<223> G

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ vskip1.000000 \ baselineskip
      

13

Claims (10)

1. Procedure for detecting the presence of or predisposition to hypercholesterolemia, disorders of lipid and lipoprotein metabolism, atherosclerosis or cardiovascular diseases in a subject, the method comprising detecting in a sample of said subject the presence of an alteration in the PCSK9 gene or the NARC-1 polypeptide, said alteration in said gene or said polypeptide being indicative of the presence of or predisposition to hypercholesterolemia, disorders of lipid and lipoprotein metabolism, atherosclerosis or cardiovascular diseases and being selected from between the group consisting of a substitution T? in nucleotide 625 of the SEC. ID. No. 1, a substitution T → C in nucleotide 890 of SEQ. ID. No. 1, an A? substitution in nucleotide 19697 of SEQ. ID. nº: 3, a substitution of the serine residue in position 127 of the SEC. ID. No. 2 with an arginine, a substitution of the phenylalanine residue at position 216 of the SEC. ID. No. 2 with a leucine, a substitution of the isoleucine residue at position 474 of the SEC. ID. No. 2 for a valine and a combination thereof. 2. Method according to claim 1, in which said alteration reduces, modifies or suppresses the activity of NARC-1 3. Procedure according to any of the claims 1 to 2, wherein said hypercholesterolemia is the autosomal dominant hypercholesterolemia. 4. Compound selection procedure biologically active, said procedure comprising:

to)

contacting a test compound with an altered PCSK9 gene or an altered NARC-1 protein or a fragment thereof of at least 15 consecutive residues comprising an alteration, wherein said alteration reduces, modifies or suppresses the activity of NARC-1 and is selected from the group consisting of a substitution T? In nucleotide 625 of the SEC. ID. No. 1, a T → C substitution in nucleotide 890 of SEQ. ID. No. 1, an A? substitution in nucleotide 19697 of SEQ. ID. nº: 3, a substitution of the serine residue in position 127 of the SEC. ID. No. 2 with an arginine, a substitution of the phenylalanine residue at position 216 of the SEC. ID. No. 2 with a leucine, a substitution of the isoleucine residue at position 474 of the SEC. ID. No. 2 for a valine and a combination thereof, and

b)

determine the capacity of said test compound to modulate the expression and / or activity of said gene or said protein or said fragment.

5. Isolated or recombinant PCSK9 gene or a fragment thereof of at least 15 consecutive nucleotides comprising an alteration, wherein said alteration is selected from the group consisting of a T → substitution in nucleotide 625 of SEQ. ID. No. 1, a T → C substitution in nucleotide 890 of SEQ. ID. No. 1, an A? substitution in nucleotide 19697 of SEQ. ID. nº: 3 and a combination thereof. 6. Isolated NARC-1 protein or purified or a fragment thereof of at least 8 amino acids consecutive comprising an alteration, in which said alteration is selected from the group consisting of a replacement of the serine residue at position 127 of the SEC. ID. nº 2 by an arginine, a substitution of the phenylalanine residue in the position 216 of the SEC. ID. No. 2 for a leucine, a substitution of the isoleucine residue at position 474 of the SEC. ID. No. 2 for one valine and a combination thereof. 7. Isolated NARC-1 protein or purified or a fragment thereof according to claim 6, in which said alteration is selected from the group constituted by a substitution of the serine residue at position 127 with a arginine, a substitution of the phenylalanine residue at position 216 for a leucine and a combination thereof. 8. Genotyped procedure of at least one polymorphism included in Table 2 which comprises (i) providing a sample of the subject and (ii) determine the identity of the allele of said polymorphism in said sample, said polymorphism being selected from the group consisting of a substitution T → in nucleotide 625 of SEQ. ID. No. 1, one T → C substitution in nucleotide 890 of SEQ. ID. nº 1, an A? Substitution in nucleotide 19697 of the SEC. ID. nº: 3 and a combination thereof, or among the group constituted by a substitution of the serine residue at position 127 of the SEC. ID. nº 2 by an arginine, a substitution of the rest phenylalanine at position 216 of the SEC. ID. No. 2 for a leucine, a replacement of the isoleucine moiety at position 474 of the SEC. ID. No. 2 for a valine and a combination thereof. 9. Diagnostic kit comprising primers and / or probes to detect in a sample of a subject the presence of an alteration in the PCSK9 gene, in which said probes or primers comprise a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length and specifically hybridize under severe conditions to an area of a PCSK9 gene that carries said alteration and said alteration is selected from the group consisting of a T → substitution in nucleotide 625 of the SEC. ID. No. 1, a T → C substitution in nucleotide 890 of SEQ. ID. No. 1, an A? substitution in nucleotide 19697 of SEQ. ID. nº: 3. 10. Kit according to claim 9, wherein said primers comprise a nucleic acid molecule single stranded 5 to 60 nucleotides in length.